Combination Of Phosphorylation Research Articles

GSK-3: Functional Insights from Cell Biology and Animal Models

Glycogen synthase kinase-3 (GSK-3) is a widely expressed and highly conserved serine/threonine protein kinase encoded in mammals by two genes that generate two related proteins: GSK-3α and GSK-3β. GSK-3 is active in cells under resting conditions and is primarily regulated through inhibition or diversion of its activity. While GSK-3 is one of the few protein kinases that can be inactivated by phosphorylation, the mechanisms of GSK-3 regulation are more varied and not fully understood. Precise control appears to be achieved by a combination of phosphorylation, localization, and sequestration by a number of GSK-3-binding proteins. GSK-3 lies downstream of several major signaling pathways including the phosphatidylinositol 3′ kinase pathway, the Wnt pathway, Hedgehog signaling and Notch. Specific pools of GSK-3, which differ in intracellular localization, binding partner affinity, and relative amount are differentially sensitized to several distinct signaling pathways and these sequestration mechanisms contribute to pathway insulation and signal specificity. Dysregulation of signaling pathways involving GSK-3 is associated with the pathogenesis of numerous neurological and psychiatric disorders and there are data suggesting GSK-3 isoform-selective roles in several of these. Here, we review the current knowledge of GSK-3 regulation and targets and discuss the various animal models that have been employed to dissect the functions of GSK-3 in brain development and function through the use of conventional or conditional knockout mice as well as transgenic mice. These studies have revealed fundamental roles for these protein kinases in memory, behavior, and neuronal fate determination and provide insights into possible therapeutic interventions.

Overexpression of puromycin sensitive aminopeptidase (PSA/NPEPPS) reduces soluble tau and delays development of neuropathology in PSA/TAU-P301L double transgenic mice

Results: We found that pseudophosphorylated tau aggregates in cells when Thr 212 is mutated to Glu (Ps-tau), suggesting that phosphorylation at this site facilitates tau self-assembly. The expression of tau pseudophosphorylated at Thr212, Thr231, and Ser262 disrupts the microtubules when co-transfected with fluorescent tubulin. EB-1 is a protein that binds to the growing end of the microtubules. Cells stably transfected with fluorescent EB-1 are used to visualize the dynamics of these filaments in the live cells. These cells were transfected with an inducible system to express tau upon withdraw of doxycycline (TET-off). The effect of Ps-tau on these cells was also studied. Conclusions: These findings suggest that tau phosphorylation at Thr 212 facilitates tau self-aggregation, and that the combination of phosphorylation at Thr212, 231 and Ser262 in the same tau molecule can trigger toxic reaction.

Aurora B Phosphorylates Spatially Distinct Targets to Differentially Regulate the Kinetochore-Microtubule Interface

Accurate chromosome segregation requires carefully regulated interactions between kinetochores and microtubules, but how plasticity is achieved to correct diverse attachment defects remains unclear. Here we demonstrate that Aurora B kinase phosphorylates three spatially distinct targets within the conserved outer kinetochore KNL1/Mis12 complex/Ndc80 complex (KMN) network, the key player in kinetochore-microtubule attachments. The combinatorial phosphorylation of the KMN network generates graded levels of microtubule-binding activity, with full phosphorylation severely compromising microtubule binding. Altering the phosphorylation state of each protein causes corresponding chromosome segregation defects. Importantly, the spatial distribution of these targets along the kinetochore axis leads to their differential phosphorylation in response to changes in tension and attachment state. In total, rather than generating exclusively binary changes in microtubule binding, our results suggest a mechanism for the tension-dependent fine-tuning of kinetochore-microtubule interactions.

Crystal Structure of a Yeast Aquaporin at 1.15 Å Reveals a Novel Gating Mechanism

Author SummaryAll living organisms must regulate precisely the flow of water into and out of cells in order to maintain cell shape and integrity. Proteins of one family, the aquaporins, are found in virtually every living organism and play a major role in maintaining water homeostasis by acting as regulated water channels. Here we describe the first crystal structure of a yeast aquaporin, Aqy1, at 1.15 Å resolution, which represents the highest resolution structural data obtained to date for a membrane protein. Using this structural information, we address an outstanding biological question surrounding yeast aquaporins: what is the functional role of the amino-terminal extension that is characteristic of yeast aquaporins? Our structural data show that the amino terminus of Aqy1 fulfills a novel gate-like function by folding to form a cytoplasmic helical bundle with a tyrosine residue entering the water channel and occluding the cytoplasmic entrance. Molecular dynamics simulations and functional studies in combination with site-directed mutagenesis suggest that water flow is regulated through a combination of mechanosensitive gating and post-translational modifications such as phosphorylation. Our study therefore provides insight into a unique mechanism for the regulation of water flux in yeast.

Regulation of gene expression during M-G1-phase in fission yeast through Plo1p and forkhead transcription factors

In fission yeast the expression of several genes during M-G1 phase is controlled by binding of the PCB binding factor (PBF) transcription factor complex to Pombe cell cycle box (PCB) promoter motifs. Three components of PBF have been identified, including two forkhead-like proteins Sep1p and Fkh2p, and a MADS-box-like protein, Mbx1p. Here, we examine how PBF is controlled and reveal a role for the Polo kinase Plo1p. plo1(+) shows genetic interactions with sep1(+), fkh2(+) and mbx1(+), and overexpression of a kinase-domain mutant of plo1 abolishes M-G1-phase transcription. Plo1p binds to and directly phosphorylates Mbx1p, the first time a Polo kinase has been shown to phosphorylate a MADS box protein in any organism. Fkh2p and Sep1p interact in vivo and in vitro, and Fkh2p, Sep1p and Plo1p contact PCB promoters in vivo. However, strikingly, both Fkh2p and Plo1p bind to PCB promoters only when PCB-controlled genes are not expressed during S- and G2-phase, whereas by contrast Sep1p contacts PCBs coincident with M-G1-phase transcription. Thus, Plo1p, Fkh2p and Sep1p control M-G1-phase gene transcription through a combination of phosphorylation and cell-cycle-specific DNA binding to PCBs.

Transcription factor cAMP response element‐binding protein CREB

The cAMP response element-binding protein (CREB) was identified almost 20 years ago. Since then, CREB has become one of the most extensively studied transcription factors. Currently, the importance of CREB for numerous physiological events has been confirmed by the number of targets: up to 4000 genes or up to 6000 loci. In addition, it is well known that numerous signals, not only the cAMP signal, but also Ca, cellular stresses and cell cycle systems, regulate CREB activity in a phosphorylation-dependent manner. In mice, knockout models targeting CREB and its cognate factors have disclosed essential functions of the CREB family in cell survival from early embryo to germ cells. By contrast, the CREB family factors are found only in multicellular animals, although cAMP⁄ stressdependent gene regulation plays an important role in a variety of organisms, such as Escherichia coli and yeast, indicating that CREB may regulate physiological events acquired by animals during their evolution. In view of these findings, we summarize a new insight of CREB regulation actualized through its basic leucine zipper domain and three physiological events regulated by CREB: ischemia, tumorigenesis and memory. Hiroshi Takemori, Junko Kajimura and Mitsuhiro Okamoto describe transactivation activities encoded by the basic leucine zipper (bZIP) domain. Studies of protein–protein interaction suggest the presence of CREB’s transactivation activities outside of its transactivation domain. This notion was confirmed by the discovery of the CREB specific coactivator: transducer of regulated CREB activity (TORC). TORCs are also regulated by phosphorylation, but they are active in their dephospho-forms. The dephosphorylation of TORCs occurs concomitantly with the phosphorylation of CREB. The combination of phosphorylation and dephosphorylation of CREB and TORC allows CREB to regulate CREdependent transcription in a variety of spectra. Kazuo Kitagawa reports on the importance of CREB and CRE-mediated gene expression after ischemic stresses. When nerve cells are subjected to sublethal conditions, kinase cascades increase CREB phoshorylation and upregulate CRE-mediated expression of genes, such as Bcl-2 and brain derived neurotrophic factor, which enhance cell survival. Recent studies also provide evidence that CREB agonists, inhibitors of phosphodiesterase and acetylcholinesterase, protect neurons from stress, thus indicating the neuro-protective function of CREB. Hao Wu, Yang Zhou and Zhi-Qi Xiong deal with a mechanism by which CREB maintains memory and learning in the brain. Neuronal actions ⁄ signals are classified into two categories, nongenomic actions and genomic actions. The latter occurs in the late phase of signal transduction, and memory is established through long-term synaptic potentiation. However, CREB phosphorylation is mediated by nongenomic actions that occur in the acute phase and diminish in the late phase. TORC1 is abundantly expressed in the hippocampus and plays an important role in the prolonged activation of CREB under the regulation of cAMP ⁄Ca signaling. Yeung-Tung Siu and DongYan Jin describe the involvement of CREB in oncogenesis. Escape from cell death is an essential mechanism for cells to become immortalized. Excessive and prolonged activation of CREB then maintains cell proliferation. Some of the gene-realignments that produce chimeric proteins of oncogenic factors with CREB and its related factors induce constitutive activation of CRE-dependent transcription followed by onsets of tumorigensis. Viruses also utilize CREB for their transcription and replication, and some viral proteins consistently keep CREB activated, which then becomes a factor in virus-mediated tumorigensis. The identification and clarification of the physiological roles of CREB and its regulatory mechanisms disclosed by these studies can be expected to lead to an acceleration in related discoveries in the not too distant future.

Site-specific Phosphorylation and Caspase Cleavage Differentially Impact Tau-Microtubule Interactions and Tau Aggregation

The microtubule-associated protein tau is hyperphosphorylated and forms neurofibrillary tangles in Alzheimer disease. Additionally caspase-cleaved tau is present in Alzheimer disease brains co-localized with fibrillar tau pathologies. To further understand the role of site-specific phosphorylation and caspase cleavage of tau in regulating its function, constructs of full-length tau (T4) or tau truncated at Asp421 (T4C3) to mimic caspase-3 cleavage with and without site-directed mutations that mimic phosphorylation at Thr231/Ser235, Ser396/Ser404, or at all four sites (Thr231/Ser235/Ser396/Ser404) were made and expressed in cells. Pseudophosphorylation of T4, but not T4C3, at either Thr231/Ser235 or Ser396/Ser404 increased its phosphorylation at Ser262 and Ser199. Pseudophosphorylation at Thr231/Ser235 impaired the microtubule binding of both T4 and T4C3. In contrast, pseudophosphorylation at Ser396/Ser404 only affected microtubule binding of T4C3 but did make T4 less soluble and more aggregated, which is consistent with the previous finding (Abraha, A., Ghoshal, N., Gamblin, T. C., Cryns, V., Berry, R. W., Kuret, J., and Binder, L. I. (2000) J. Cell Sci. 113, 3737-3745) that pseudophosphorylation at Ser396/Ser404 enhances tau polymerization in vitro. In situ T4C3 was more prevalent in the cytoskeletal and microtubule-associated fractions compared with T4, whereas purified recombinant T4 bound microtubules with higher affinity than did T4C3 in an in vitro assay. These data indicate the importance of cellular factors in regulating tau-microtubule interactions and that, in the cells, phosphorylation of T4 might impair its microtubule binding ability more than caspase cleavage. Treatment of cells with nocodazole revealed that pseudophosphorylation of T4 at both Thr231/Ser235 and Ser396/Ser404 diminished the ability of tau to protect against microtubule depolymerization, whereas with T4C3 only pseudophosphorylation at Ser396/Ser404 attenuated the ability of tau to stabilize the microtubules. These results show that site-specific phosphorylation and caspase cleavage of tau differentially affect the ability of tau to bind and stabilize microtubules and facilitate tau self-association.

The Low Density Lipoprotein Receptor-related Protein 6 Interacts with Glycogen Synthase Kinase 3 and Attenuates Activity

Glycogen synthase kinase 3 (GSK3) is a widely expressed Ser/Thr protein kinase that phosphorylates numerous substrates. This large number of substrates requires precise and specific regulation of GSK3 activity, which is achieved by a combination of phosphorylation, localization, and interactions with GSK3-binding proteins. Members of the Wnt canonical pathway have been shown to influence GSK3 activity. Through a yeast two-hybrid screen, we identified the Wnt canonical pathway co-receptor protein low density lipoprotein receptor-related protein 6 (LRP6) as a GSK3-binding protein. The interaction between the C terminus of LRP6 and GSK3 was also confirmed by in vitro GST pull-down assays and in situ coimmunoprecipitation assays. In vitro assays using immunoprecipitated proteins demonstrated that the C terminus of LRP6 significantly attenuated the activity of GSK3beta. In situ, LRP6 significantly decreased GSK3beta-mediated phosphorylation of tau at both primed and unprimed sites. Finally, it was also demonstrated that GSK3beta phosphorylates the PPP(S/T)P motifs in the C terminus of LRP6. This is the first identification of a direct interaction between LRP6 and GSK3, which results in an attenuation of GSK3 activity.

Growth factors induce differential phosphorylation profiles of the Hrs–STAM complex: a common node in signalling networks with signal-specific properties

Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) and STAM (signal-transducing adaptor molecule) form a heterodimeric complex that associates with endosomal membranes and is tyrosine-phosphorylated in response to a variety of growth factors including EGF (epidermal growth factor), HGF (hepatocyte growth factor) and PDGF (platelet-derived growth factor). Phosphorylation of the Hrs-STAM complex requires receptor endocytosis. We show that an intact UIM (ubiquitin interaction motif) within Hrs is a conserved requirement for Hrs phosphorylation downstream of both EGF and HGF stimulations. Consistent with this, expression of a dominant-negative form of the E3 ubiquitin ligase, c-Cbl, inhibits EGF- and HGF-dependent Hrs phosphorylation. Despite this conservation, kinase inhibitor profiles using PP1 (4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) and SU6656 indicate that distinct non-receptor tyrosine kinases couple EGF, HGF and PDGF stimulation with the tyrosine phosphorylation of the Hrs-STAM complex. Crucially, analysis with phospho-specific antibodies indicates that these kinases generate a signal-specific, combinatorial phosphorylation profile of the Hrs-STAM complex, with the potential of diversifying tyrosine kinase receptor signalling through a common element.

Cdc25A phosphatase: combinatorial phosphorylation, ubiquitylation and proteolysis.

In eukaryotic cells, control mechanisms of cell-cycle progression have evolved to accurately monitor the integrity of genetic information to be transferred to the progeny. Cdc25A phosphatase is an essential activator of cell-cycle progression and is targeted by checkpoint signals. Ubiquitylation regulates Cdc25A activity through fine tuning of its protein levels. Two different ubiquitin ligases (APC/C and SCF complex) are involved in Cdc25A turnover. While APC/C is involved in regulating Cdc25A at the exit of mitosis, SCF regulates the abundance of Cdc25A in S phase and G2. In response to DNA damage or to stalled replication, the activation of the ATM and ATR protein kinases leads to Chk1 and Chk2 activation and to Cdc25A hyperphosphorylation. These events stimulate SCF-mediated ubiquitylation of Cdc25A and its proteolysis. This contributes to delaying cell-cycle progression, thereby preventing genomic instability. Based on recent findings, we discuss the role of Cdc25A ubiquitylation and degradation in cell-cycle progression and in response to DNA damage. Moreover, we discuss the role of phosphorylation at multiple sites in triggering ubiquitylation signals.

Cloning of a Human Type II Phosphatidylinositol 4-Kinase Reveals a Novel Lipid Kinase Family

Phosphoinositide lipids regulate numerous cellular processes in all eukaryotes. The versatility of this phospholipid is provided by combinations of phosphorylation on the 3', 4', and 5' positions of the inositol head group. Two distinct structural families of phosphoinositide (PI) kinases have so far been identified and named after their prototypic members, the PI 3-kinase and phosphatidylinositol (PtdIns) phosphate kinase families, both of which have been found to contain structural homologues possessing PI 4-kinase activity. Nevertheless, the prevalent PtdIns 4-kinase activity in many mammalian cell types is conferred by the widespread type II PtdIns 4-kinase, which has so far resisted molecular characterization. We have partially purified the human type II isoform from plasma membrane rafts of human A431 epidermoid carcinoma cells and obtained peptide mass and sequence data. The results allowed the cDNA containing the full open reading frame to be cloned. The predicted amino acid sequence revealed that the type II enzyme is the prototypic member of a novel, third family of PI kinases. We have named the purified protein type IIalpha and a second human isoform, type IIbeta. The type IIalpha mRNA appears to be expressed ubiquitously in human tissues, and homologues appear to be expressed in all eukaryotes.

Modulating nuclear receptor function: may the phos be with you.

Gene expression is tightly regulated in response to both extracellular and intracellular signals, which control virtually every biological process. Intracellular signaling molecules such as steroid hormone, thyroid hormone, retinoids, and vitamin D exert their function through direct binding to their cognate nuclear receptors (1). Nuclear receptors are transcription factors that recognize specific sequences in target genes via a centrally located DNA-binding domain (DBD). Some receptors bind DNA as monomers, some as homodimers, and some as heterodimers with a common partner, the retinoid X receptor (RXR). Both the DBD and the COOH-terminal ligand-binding domain (LBD) contribute to dimerization. Many nuclear receptors repress transcription in the absence of ligand, due to a repression function in the LBD. Interaction of the LBD with ligand abolishes repression and activates transcription via a COOH-terminal activation domain (activation function 2, or AF2), which in some receptors works in tandem with an additional activation domain (AF1) in the NH2-terminus, or A/B region. There are over 150 mammalian members of the nuclear receptor superfamily. Many of these are orphan receptors, whose ligands are yet to be discovered. In contrast to the small lipophilic molecules that signal via nuclear receptors, extracellular signaling molecules such as peptide hormones and cytokines communicate with their intracellular targets through surface receptors, which activate signal transduction pathways that ultimately lead to regulation of gene expression mediated by transcription factors such as fos, jun, cAMP-response element binding protein (CREB), and others. Most often the mechanism involves direct phosphorylation of the transcription factor by a kinase that is activated as a result of the ligand-receptor interaction at the cell surface. Nuclear receptors are also targets of kinases involved in signal transduction. In this issue of the JCI, Kremer and colleagues report that activation of mitogen-activated protein kinase (MAPK) attenuates the ligand-dependent transactivation function of the vitamin D receptor (VDR) and its heterodimer partner RXR (2). This effect appears to be mediated by phosphorylation of human RXRα on a serine residue at amino acid 260 in the protein. This report not only provides a potential mechanism for ras-induced transformation of human keratinocytes, but also has broader implications because RXR heterodimerizes with several other nuclear receptors including thyroid hormone receptor, retinoic acid receptor, and peroxisome proliferator–activated receptor. Thus, it will be interesting to determine in future studies whether phosphorylation of RXR alters the biological activity of these receptors. Phosphorylation of nuclear receptors provides an important mechanism for crosstalk between signaling pathways. Phosphorylation has been shown to modulate the activity of many nuclear receptors (Table ​(Table1).1). Multiple kinase pathways have been implicated, including cAMP-dependent protein kinase, casein kinase, glycogen synthase kinase (GSK), jun kinase, cyclin-dependent kinases (Cdks), and MAPKs. All aspects of receptor function can be regulated, including DNA binding and dimerization, transcriptional activity, interaction with cofactors, and ligand-binding affinity. Table 1 Phosphorylation of nuclear receptors The mechanism by which phosphorylation of RXR inhibits vitamin D signaling is not clear. Phosphorylated RXR was not recognized by an antibody that recognized the nonphosphorylated protein, suggesting that phosphorylation resulted in a conformational change in the protein. Such a change in tertiary structure might alter affinity of the VDR/RXR heterodimer for transcriptional coregulators. This is an attractive hypothesis because ligand activation of nuclear receptors is clearly related to a conformational change that favors coactivator interaction (3). An alternative possibility is that the phosphate group has a more direct role in a critical function such as coregulator interaction, as has been observed for the interaction between phosphorylated CREB and its main coactivator CBP (4). Although the structures of the DBD and LBD have been solved for numerous receptors, no full-length receptor has been crystallized, nor has the structure of any phosphorylated nuclear receptor been determined. Thus, it remains to be determined which effects of phosphorylation listed in Table ​Table11 are due to conformational changes and which are due to more localized effects of the phosphate group on receptor function. Moreover, the combination of phosphorylation and ligand binding may in some cases lead to an entirely novel structure. The concept that nuclear hormone receptor function is determined by rapid, reversible, and combinatorial structural changes governing interactions with other cellular proteins provides a framework for understanding how the receptor can integrate a variety of signals in a physiological context. This level of understanding puts us light years ahead of where we were just a short time ago.

Serine 16 of Stathmin as a Cytosolic Target for Ca2+/Calmodulin-Dependent Kinase II After CD2 Triggering of Human T Lymphocytes

We investigated specific signaling events initiated after T cell triggering through the costimulatory surface receptors CD2 and CD28 as compared with activation via the Ag receptor (TCR/CD3). We therefore followed the phosphorylation of stathmin, a ubiquitous cytoplasmic phosphoprotein proposed as a general relay integrating diverse intracellular signaling pathways through the combinatorial phosphorylation of serines 16, 25, 38, and 63, the likely physiologic substrates for Ca2+/calmodulin (CaM)-dependent kinases, mitogen-activated protein (MAP) kinase, cyclin-dependent kinases (cdks), and protein kinase A, respectively. We addressed the specific protein kinase systems involved in the CD2 pathway of T cell activation through the analysis of stathmin phosphorylation patterns in exponentially growing Jurkat T cells, as revealed by phosphopeptide mapping. Stimulation via CD2 activated multiple signal transduction pathways, resulting in phosphorylation of distinct sites of stathmin, the combination of which only partially overlaps the CD3- and CD28-induced patterns. The partial redundancy of the three T cell activation pathways was evidenced by the phosphorylation of Ser25 and Ser38, substrates of MAP kinases and of the cdk family kinase(s), respectively. Conversely, the phosphorylation of Ser16 of stathmin was observed in response to both CD2 and CD28 triggering, but not CD3 triggering, with a kinetics compatible with the lasting activation of CaM kinase II in response to CD2 triggering. In vitro, Ser16 of recombinant human stathmin was phosphorylated also by purified CaM kinase II, and in vivo, CaM kinase II activity was indeed stimulated in CD2-triggered Jurkat cells. Altogether, our results favor an association of CaM kinase II activity with costimulatory signals of T lymphocyte activation and phosphorylation of stathmin on Ser16.